Laboratory Investigation (2013) 93, 701–710 & 2013 USCAP, Inc All rights reserved 0023-6837/13 Evaluating the repair of DNA derived from formalin-fixed paraffin-embedded tissues prior to genomic profiling by SNP–CGH analysis Abdel Nasser Hosein1,*, Sarah Song2,3,*, Amy E McCart Reed3, Janani Jayanthan3, Lynne E Reid3, Jamie R Kutasovic3, Margaret C Cummings3,4, Nic Waddell2, Sunil R Lakhani3,4,5, Georgia Chenevix-Trench1 and Peter T Simpson3 Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples. Owing to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromized, which means obtaining informative data regarding epigenetic, genomic, and expression altera- tions can be challenging. Here, we have investigated the utility of repairing damaged DNA derived from FFPE tumors prior to single-nucleotide polymorphism (SNP) arrays for whole-genome DNA copy number analysis. DNA was extracted from FFPE samples spanning five decades, involving tumor material obtained from surgical specimens and postmortems. Various aspects of the protocol were assessed, including the method of DNA extraction, the role of Quality Control quantitative PCR (qPCR) in predicting sample success, and the effect of DNA restoration on assay performance, data quality, and the prediction of copy number aberrations (CNAs). DNA that had undergone the repair process yielded higher SNP call rates, reduced log R ratio variance, and improved calling of CNAs compared with matched FFPE DNA not subjected to repair. Reproducible mapping of genomic break points and detection of focal CNAs representing high- level gains and homozygous deletions (HD) were possible, even on autopsy material obtained in 1974. For example, DNA amplifications at the ERBB2 and EGFR gene loci and a HD mapping to 13q14.2 were validated using immuno- histochemistry, in situ hybridization, and qPCR. The power of SNP arrays lies in the detection of allele-specific aberrations; however, this aspect of the analysis remains challenging, particularly in the distinction between loss of heterozygosity (LOH) and copy neutral LOH. In summary, attempting to repair DNA that is damaged during fixation and storage may be a useful pretreatment step for genomic studies of large archival FFPE cohorts with long-term follow-up or for understanding rare cancer types, where fresh frozen material is scarce. Laboratory Investigation (2013) 93, 701–710; doi:10.1038/labinvest.2013.54; published online 8 April 2013 KEYWORDS: array CGH; breast cancer; DNA restoration; DNA copy number alterations; formalin fixed, paraffin-embedded tissue; SNP array Archived formalin-fixed paraffin-embedded (FFPE) tumors modified during formalin fixation, and as a result of the long represent a rich reservoir of tissue samples for cancer re- storage times. Nevertheless, several molecular assays have search. Being able to access this resource to obtain high- been developed that can tolerate low-quality nucleic acids to quality molecular data is important for investigating the generate genomic or expression data that is informative to biology underlying rare tumor types, complex malignant tumor biology. processes, such as metastatic progression, and long-term Chromosomal genomic hybridization (CGH), first de- clinical outcome studies. Molecular analyses of FFPE sam- scribed over two decades ago,1 is a cytogenetic technique that ples, however, is extremely challenging, as the DNA and RNA interrogates chromosomal imbalances across the genome.2 It isolated from such samples is often degraded and chemically has been instrumental in defining genome-wide DNA copy 1Cancer Genetics Laboratory, Queensland Institute of Medical Research, Herston QLD, Australia; 2Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia; 3The University of Queensland, UQ Centre for Clinical Research, Royal Brisbane and Women’s Hospital, Herston, Brisbane, QLD, Australia; 4Pathology Queensland, Royal Brisbane and Women’s Hospital, Brisbane, QLD, Australia and 5The University of Queensland, School of Medicine, Brisbane, QLD, Australia Correspondence: Dr PT Simpson, PhD, UQ Centre for Clinical Research, The University of Queensland, Building 71/918, Royal Brisbane and Women’s Hospital, Herston, QLD 4029, Australia. E-mail: [email protected] *These authors contributed equally to this work. Received 10 January 2013; revised 25 February 2013; accepted 28 February 2013 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 93 June 2013 701 FFPE DNA restoration for SNP–CGH analysis AN Hosein et al number changes that occur in human cancers and can readily MATERIALS AND METHODS tolerate fragmented DNA derived from FFPE tissue. Early Clinical Cohort array-based CGH (aCGH) platforms were dependent on Ethical approval for the research was obtained from the bacterial artificial chromosomes containing large fragments Human Research Ethics Committees of the Royal Brisbane of the human genome3–6 but have been largely supplanted by and Women’s Hospital (RBWH) and The University commercially available platforms utilizing long of Queensland. The biospecimens used in this study were oligonucleotide-based or single-nucleotide polymorphism obtained from the RBWH pathology department or from the (SNP) markers as probes.7–10 Brisbane Breast Bank, and involved archival FFPE tissue Initially designed for use in genetic association studies, samples, FF tumor tissue, and DNA from blood. In total, SNP arrays have also furthered our understanding of struc- tissue specimens from 25 cases were used, encompassing tural variation in cancer. SNP arrays (eg, from vendors such (i) surgical tissue samples (tumor and matched normal DNA as Affymetrix or Illumina) are the platform of choice by large from blood) from two cases diagnosed in 2010, (ii) surgical consortia such as the International Cancer Genome Con- tissue samples (tumor and matched normal) from five cases sortium (ICGC), The Cancer Genome Atlas (TCGA), and diagnosed between 1987 and 1990, and (iii) tissue samples Molecular Taxonomy of Breast Cancer International Con- taken during autopsies performed on 18 patients who died sortium (METABRIC)11–13 for studying somatic DNA copy from metastatic breast cancer between 1959 and 2001. An number alterations in fresh frozen (FF) tumor samples, as, overview of the specimens and the analyses performed are unlike aCGH platforms, they are also capable of detecting outlined in Table 1, with further experimental details given in copy neutral loss of heterozygosity (LOH) events. There are, Supplementary Table 1. Notably, case Q590 had tumor DNA however, limited data demonstrating the tolerance of this from both an FFPE block and a matched FF piece of tissue; technology to compromized DNA quality derived from large for cases Q590 and 007, the tumor DNA was analyzed by series of FFPE tumors. Comparative analyses of matched SNP–CGH with and without undergoing the restoration pairs of FF and FFPE tumors demonstrate that SNP array process; and for five cases (007, 261, 276, 318, 540), the DNA technology can yield reasonable DNA copy number data.14–19 was extracted using two different DNA extraction methods to However, reduced data quality obtained from FFPE samples see if this affected data quality. on SNP arrays can lead to the false detection of copy number aberrations (CNAs) that are not identified using DNA Extraction oligonucleotide aCGH platforms or using matched FF For all FFPE samples, tumor-rich areas were identified from a tumors on SNP-based microarrays.20 Oligonucleotide freshly cut hematoxylin and eosin-stained section, and cores aCGH platforms developed by Agilent or Nimblegen are of tumor tissue were punched using a 1-mm diameter tissue thus considered as the more robust at tolerating degraded microarray needle. Cores were dewaxed and rehydrated DNA from FFPE tissue samples than SNP arrays. They according to the standard protocols. Two methods of DNA provide both high resolution and precision for detecting extraction were used: the DNeasy Blood and Tissue Kit high-level amplifications, single-copy alterations, and (Qiagen Pty, Chadstone, VIC, Australia) and the High Pure resolving chromosomal break points,20,21 but lack the DNA Template Preparation Kit (Roche Australia Pty, Castle capacity for detecting copy neutral LOH. Hill, NSW, Australia), which was recommended by Illumina. Several technical developments have been introduced that Both were performed according to the manufacturer’s in- may help optimize performance of FFPE-derived DNA in SNP structions with the following exceptions for both techniques: array analysis. For example, the introduction of a prequalifier (i) some tissue samples (Supplementary Table 1) were pre- PCR step provides a means of predicting which FFPE DNA treated in 1 M sodium thiocyanate overnight at 37 1Cto samples might yield sufficient quality SNP array data.15,20,22 remove crosslinks and (ii) for all cases an extended tissue The recently developed Oncoscan FFPE platform (Affymetrix), digestion step was performed over three nights with sup- a high-resolution SNP array based on molecular inversion plementary Proteinase K (Invitrogen) added every 24 h. probes, appears to perform well in comparison with aCGH Eluted DNA was assessed for purity using the Nanodrop- platforms and so might prove to be a successful method for 2000, and double-stranded DNA was quantified